ABSTRACT: Our goal is leverage our recent insights into coronavirus B conserved RNA structures, and our discovery of formulations for high efficiency lung delivery, into the rapid development of SARS-CoV-2 specific therapeutics. Using a novel suite of computational technology tools, we have identified predicted RNA secondary structures in regions conserved across coronavirus B genomes including SARS-CoV-2. We have also identified two tandem predicted microRNA 191 (miR191) binding sites within the 5?-most such structure. In our current grant on influenza A virus (IAV), we identified an RNA secondary structure conserved across all IAV isolates that is essential for in vitro packaging and in vivo disease, then designed short highly stable locked nucleic acid (LNA) oligonucleotides to bind and distort this RNA packaging signal, and demonstrated that a single dose of our lead LNA can a) provide immediate 100% protection for over 14 days from a lethal inoculum of IAV, b) provide 100% survival when administered 3 days after a lethal IAV inoculum, and c) while sufficiently attenuating the infection, enable the subsequent development of high level immunity. Moreover, we have also recently discovered that empty deproteinized pollen shells represent an outstanding vehicle for delivery of LNAs to the lung with much greater efficacy and tolerability than current formulations for nucleic acid delivery. We now hypothesize that 1) our identified RNA secondary structures in SARS-CoV-2 represent ideal candidate targets for disrupting the virus lifecycle, via structure-specific LNAs; 2) the miR191 binding sites within the 5?-most conserved RNA secondary structure reflect an essential mechanism for regulating translation of corona B viruses that is amenable to targeting by specifically designed LNAs; 3) our novel deproteinized pollen formulation represents an ideal means of delivering such LNAs to both prevent and treat established SARS-CoV-2 infections. We will test these hypotheses via the following specific aims that are to: 1) Determine which LNA gapmers from a screening panel synthesized against our identified conserved RNA secondary structure targets are most disruptive to the latter?s integrity, as assessed by SHAPE, REVI, and Mutate-and-Map; 2) Refine the sequence (total LNA length, fine nucleotide target position, and length of single stranded DNA gapmer) of the top performing LNA and test a panel of LNA analogs to identify the most potent disrupter of targeted SARS-CoV-2 conserved RNA secondary structure; 3) Determine the effect of LNAs designed to sequester miR191 in cells transfected with a SARS-CoV-2 5? terminal RNA segment linked to a luciferase reporter; 4) Determine the effect of the identified lead LNAs (targeting conserved SARS-CoV-2 RNA secondary structure, and sequestering miR191) on cells infected with SARS-CoV-2 in vitro, and in vivo when delivered intranasally by current lung-targeting transfection reagent (i.e.JetPEi) vs. pollen shells to SARS-CoV- 2-infected mice. Successful accomplishment of our aims will yield proof-of-concept for an exciting new class of anti- SARS-CoV-2 RNA therapeutics within the short time frame of this proposal.